Overexpression of SLIM1 transcription factor accelerates vegetative development in Arabidopsis thaliana.

The transcription factor Sulfur Limitation 1 (SLIM1) belongs to the plant-specific Ethylene Insenstive3-Like transcription factor family and is known to coordinate gene expression in response to sulfur deficiency. However, the roles of SLIM1 in nutrient-sufficient conditions have not been characterized. Employing constitutive SLIM1 overexpression (35S::SLIM1) and CRISPR/Cas9 mutant plants (slim1-cr), we identified several distinct phenotypes in nutrient-sufficient conditions in Arabidopsis thaliana. Overexpression of SLIM1 results in plants with approximately twofold greater rosette area throughout vegetative development. 35S::SLIM1 plants also bolt earlier and exhibit earlier downregulation of photosynthesis-associated genes and earlier upregulation of senescence-associated genes than Col-0 and slim1-cr plants. This suggests that overexpression of SLIM1 accelerates development in A. thaliana. Genome-wide differential gene expression analysis relative to Col-0 at three time points with slim1-cr and two 35S::SLIM1 lines allowed us to identify 1,731 genes regulated directly or indirectly by SLIM1 in vivo.

Short wind pulses consistently change the morphology of roots, but not of shoots, across young plants of different growth forms.

Wind is an environmental stimulus that stresses plants of all growth forms at all life-stages by influencing the development, architecture, and morphology of roots and shoots. However, comparative studies are scarce and no study directly investigated whether shoot and root morphological traits of trees, grasses and forbs differ in their response to short wind pulses of different wind intensity. In this study, we found that across species, wind stress by short wind pulses of increasing intensity consistently changed root morphology, but did not affect shoot morphological traits, except plant height in four species. Wind effects in roots were generally weak in tree species but consistent across growth forms. Furthermore, plant height of species was correlated with changes in specific root length and average diameter.Our results indicate that short-pulse wind treatments affect root morphology more than shoot morphology across growth forms. They further suggest that wind stress possibly promotes root anchorage in young plants and that these effects might depend on plant height.

Arabidopsis BBX14 negatively regulates nitrogen starvation- and dark-induced leaf senescence.

Senescence is a highly regulated process driven by developmental age and environmental factors. Although leaf senescence is accelerated by nitrogen (N) deficiency, the underlying physiological and molecular mechanisms are largely unknown. Here, we reveal that BBX14, a previously uncharacterized BBX-type transcription factor in Arabidopsis, is crucial for N starvation-induced leaf senescence. We find that inhibiting BBX14 by artificial miRNA (amiRNA) accelerates senescence during N starvation and in darkness, while BBX14 overexpression (BBX14-OX) delays it, identifying BBX14 as a negative regulator of N starvation- and dark-induced senescence. During N starvation, nitrate and amino acids like glutamic acid, glutamine, aspartic acid, and asparagine were highly retained in BBX14-OX leaves compared to the wild type. Transcriptome analysis showed a large number of senescence-associated genes (SAGs) to be differentially expressed between BBX14-OX and wild-type plants, including ETHYLENE INSENSITIVE3 (EIN3) which regulates N signaling and leaf senescence. Chromatin immunoprecipitation (ChIP) showed that BBX14 directly regulates EIN3 transcription. Furthermore, we revealed the upstream transcriptional cascade of BBX14. By yeast one-hybrid screen and ChIP, we found that MYB44, a stress-responsive MYB transcription factor, directly binds to the promoter of BBX14 and activates its expression. In addition, Phytochrome Interacting Factor 4 (PIF4) binds to the promoter of BBX14 to repress BBX14 transcription. Thus, BBX14 functions as a negative regulator of N starvation-induced senescence through EIN3 and is directly regulated by PIF4 and MYB44.

New insights into the regulation of plant metabolism by O-acetylserine: sulfate and beyond.

Under conditions of sulfur deprivation, O-acetylserine (OAS) accumulates, which leads to the induction of a common set of six genes, called OAS cluster genes. These genes are induced not only under sulfur deprivation, but also under other conditions where OAS accumulates, such as shift to darkness and stress conditions leading to reactive oxygen species (ROS) or methyl-jasmonate accumulation. Using the OAS cluster genes as a query in ATTED-II, a co-expression network is derived stably spanning several hundred conditions. This allowed us not only to describe the downstream function of the OAS cluster genes but also to score for functions of the members of the co-regulated co-expression network and hence the effects of the OAS signal on the sulfate assimilation pathway and co-regulated pathways. Further, we summarized existing knowledge on the regulation of the OAS cluster and the co-expressed genes. We revealed that the known sulfate deprivation-related transcription factor EIL3/SLIM1 exhibits a prominent role, as most genes are subject to regulation by this transcription factor. The role of other transcription factors in response to OAS awaits further investigation.

The SLIM1 transcription factor affects sugar signaling during sulfur deficiency in Arabidopsis.

The homeostasis of major macronutrient metabolism needs to be tightly regulated, especially when the availability of one or more nutrients fluctuates in the environment. Both sulfur metabolism and glucose signaling are important processes throughout plant growth and development, as well as during stress responses. Still, very little is known about how these processes affect each other, although they are positively connected. Here, we showed in Arabidopsis that the crucial transcription factor of sulfur metabolism, SLIM1, is involved in glucose signaling during shortage of sulfur. The germination rate of the slim1_KO mutant was severely affected by high glucose and osmotic stress. The expression of SLIM1-dependent genes in sulfur deficiency appeared to be additionally induced by a high concentration of either mannitol or glucose, but also by sucrose, which is not only the source of glucose but another signaling molecule. Additionally, SLIM1 affects PAP1 expression during sulfur deficiency by directly binding to its promoter. The lack of PAP1 induction in such conditions leads to much lower anthocyanin production. Taken together, our results indicate that SLIM1 is involved in the glucose response by modulating sulfur metabolism and directly controlling PAP1 expression in Arabidopsis during sulfur deficiency stress.

In silico analysis of cis-elements and identification of transcription factors putatively involved in the regulation of the OAS cluster genes SDI1 and SDI2.

Arabidopsis thaliana sulfur deficiency-induced 1 and sulfur deficiency-induced 2 (SDI1 and SDI2) are involved in partitioning sulfur among metabolite pools during sulfur deficiency, and their transcript levels strongly increase in this condition. However, little is currently known about the cis- and trans-factors that regulate SDI expression. We aimed at identifying DNA sequence elements (cis-elements) and transcription factors (TFs) involved in regulating expression of the SDI genes. We performed in silico analysis of their promoter sequences cataloging known cis-elements and identifying conserved sequence motifs. We screened by yeast-one-hybrid an arrayed library of Arabidopsis TFs for binding to the SDI1 and SDI2 promoters. In total, 14 candidate TFs were identified. Direct association between particular cis-elements in the proximal SDI promoter regions and specific TFs was established via electrophoretic mobility shift assays: sulfur limitation 1 (SLIM1) was shown to bind SURE cis-element(s), the basic domain/leucine zipper (bZIP) core cis-element was shown to be important for HY5-homolog (HYH) binding, and G-box binding factor 1 (GBF1) was shown to bind the E box. Functional analysis of GBF1 and HYH using mutant and over-expressing lines indicated that these TFs promote a higher transcript level of SDI1 in vivo. Additionally, we performed a meta-analysis of expression changes of the 14 TF candidates in a variety of conditions that alter SDI expression. The presented results expand our understanding of sulfur pool regulation by SDI genes.

Sulfur deficiency-induced genes affect seed protein accumulation and composition under sulfate deprivation.

Sulfur deficiency-induced proteins SDI1 and SDI2 play a fundamental role in sulfur homeostasis under sulfate-deprived conditions (-S) by downregulating glucosinolates. Here, we identified that besides glucosinolate regulation under -S, SDI1 downregulates another sulfur pool, the S-rich 2S seed storage proteins in Arabidopsis (Arabidopsis thaliana) seeds. We identified that MYB28 directly regulates 2S seed storage proteins by binding to the At2S4 promoter. We also showed that SDI1 downregulates 2S seed storage proteins by forming a ternary protein complex with MYB28 and MYC2, another transcription factor involved in the regulation of seed storage proteins. These findings have significant implications for the understanding of plant responses to sulfur deficiency.

Characterization of the Heat-Stable Proteome during Seed Germination in Arabidopsis with Special Focus on LEA Proteins.

During seed germination, desiccation tolerance is lost in the radicle with progressing radicle protrusion and seedling establishment. This process is accompanied by comprehensive changes in the metabolome and proteome. Germination of Arabidopsis seeds was investigated over 72 h with special focus on the heat-stable proteome including late embryogenesis abundant (LEA) proteins together with changes in primary metabolites. Six metabolites in dry seeds known to be important for seed longevity decreased during germination and seedling establishment, while all other metabolites increased simultaneously with activation of growth and development. Thermo-stable proteins were associated with a multitude of biological processes. In the heat-stable proteome, a relatively similar proportion of fully ordered and fully intrinsically disordered proteins (IDP) was discovered. Highly disordered proteins were found to be associated with functional categories development, protein, RNA and stress. As expected, the majority of LEA proteins decreased during germination and seedling establishment. However, four germination-specific dehydrins were identified, not present in dry seeds. A network analysis of proteins, metabolites and amino acids generated during the course of germination revealed a highly connected LEA protein network.

Assessing Dynamic Changes of Taste-Related Primary Metabolism During Ripening of Durian Pulp Using Metabolomic and Transcriptomic Analyses.

Durian is an economically important fruit of Southeast Asia. There is, however, a lack of in-depth information on the alteration of its metabolic networks during ripening. Here, we annotated 94 ripening-associated metabolites from the pulp of durian cv. Monthong fruit at unripe and ripe stages, using capillary electrophoresis- and gas chromatography- time-of-flight mass spectrometry, specifically focusing on taste-related metabolites. During ripening, sucrose content increased. Change in raffinose-family oligosaccharides are reported herein for the first time. The malate and succinate contents increased, while those of citrate, an abundant organic acid, were unchanged. Notably, most amino acids increased, including isoleucine, leucine, and valine, whereas aspartate decreased, and glutamate was unchanged. Furthermore, transcriptomic analysis was performed to analyze the dynamic changes in sugar metabolism, glycolysis, TCA cycle, and amino acid pathways to identify key candidate genes. Taken together, our results elucidate the fundamental taste-related metabolism of durian, which can be exploited to develop durian metabolic and genetic markers in the future.

Developmental stage-specific metabolite signatures in Arabidopsis thaliana under optimal and mild nitrogen limitation.

Metabolites influence flowering time, and thus are among the major determinants of yield. Despite the reported role of trehalose 6-phosphate and nitrate signaling on the transition from the vegetative to the reproductive phase, little is known about other metabolites contributing and responding to developmental phase changes. To increase our understanding which metabolic traits change throughout development in Arabidopsis thaliana and to identify metabolic markers for the vegetative and reproductive phases, especially among individual amino acids (AA), we profiled metabolites of plants grown in optimal (ON) and limited nitrogen (N) (LN) conditions, the latter providing a mild but consistent limitation of N. We found that although LN plants adapt their growth to a decreased level of N, their metabolite profiles are strongly distinct from ON plant profiles, with N as the driving factor for the observed differences. We demonstrate that the vegetative and the reproductive phase are not only marked by growth parameters such as biomass and rosette area, but also by specific metabolite signatures including specific single AA. In summary, we identified N-dependent and -independent indicators manifesting developmental stages, indicating that the plant's metabolic status also reports on the developmental phases.

The Transcription Factor EIL1 Participates in the Regulation of Sulfur-Deficiency Response.

Sulfur, an indispensable constituent of many cellular components, is a growth-limiting macronutrient for plants. Thus, to successfully adapt to changing sulfur availability and environmental stress, a sulfur-deficiency response helps plants to cope with the limited supply. On the transcriptional level, this response is controlled by SULFUR LIMITATION1 (SLIM1), a member of the ETHYLENE-INSENSITIVE3-LIKE (EIL) transcription factor family. In this study, we identified EIL1 as a second transcriptional activator regulating the sulfur-deficiency response, subordinate to SLIM1/EIL3. Our comprehensive RNA sequencing analysis in Arabidopsis (Arabidopsis thaliana) allowed us to obtain a complete picture of the sulfur-deficiency response and quantify the contributions of these two transcription factors. We confirmed the key role of SLIM1/EIL3 in controlling the response, particularly in the roots, but showed that in leaves more than 50% of the response is independent of SLIM1/EIL3 and EIL1. RNA sequencing showed an additive contribution of EIL1 to the regulation of the sulfur-deficiency response but also identified genes specifically regulated through EIL1. SLIM1/EIL3 seems to have further functions (e.g. in the regulation of genes responsive to hypoxia or mediating defense at both low and normal sulfur supply). These results contribute to the dissection of mechanisms of the sulfur-deficiency response and provide additional possibilities to improve adaptation to sulfur-deficiency conditions.

Cysteine and Methionine Biosynthetic Enzymes Have Distinct Effects on Seed Nutritional Quality and on Molecular Phenotypes Associated With Accumulation of a Methionine-Rich Seed Storage Protein in Rice.

Staple crops in human and livestock diets suffer from deficiencies in certain "essential" amino acids including methionine. With the goal of increasing methionine in rice seed, we generated a pair of "Push × Pull" double transgenic lines, each containing a methionine-dense seed storage protein (2S albumin from sunflower, HaSSA) and an exogenous enzyme for either methionine (feedback desensitized cystathionine gamma synthase from Arabidopsis, AtD-CGS) or cysteine (serine acetyltransferase from E. coli, EcSAT) biosynthesis. In both double transgenic lines, the total seed methionine content was approximately 50% higher than in their untransformed parental line, Oryza sativa ssp. japonica cv. Taipei 309. HaSSA-containing rice seeds were reported to display an altered seed protein profile, speculatively due to insufficient sulfur amino acid content. However, here we present data suggesting that this may result from an overloaded protein folding machinery in the endoplasmic reticulum rather than primarily from redistribution of limited methionine from endogenous seed proteins to HaSSA. We hypothesize that HaSSA-associated endoplasmic reticulum stress results in redox perturbations that negatively impact sulfate reduction to cysteine, and we speculate that this is mitigated by EcSAT-associated increased sulfur import into the seed, which facilitates additional synthesis of cysteine and glutathione. The data presented here reveal challenges associated with increasing the methionine content in rice seed, including what may be relatively low protein folding capacity in the endoplasmic reticulum and an insufficient pool of sulfate available for additional cysteine and methionine synthesis. We propose that future approaches to further improve the methionine content in rice should focus on increasing seed sulfur loading and avoiding the accumulation of unfolded proteins in the endoplasmic reticulum. Oryza sativa ssp. japonica: urn:lsid:ipni.org:names:60471378-2.

Coordinating Sulfur Pools under Sulfate Deprivation.

Plants display manifold metabolic changes on sulfate deficiency (S deficiency) with all sulfur-containing pools of primary and secondary metabolism affected. O-Acetylserine (OAS), whose levels are rapidly altered on S deficiency, is correlated tightly with novel regulators of plant sulfur metabolism that have key roles in balancing plant sulfur pools, including the Sulfur Deficiency Induced genes (SDI1 and SDI2), More Sulfur Accumulation1 (MSA1), and GGCT2;1. Despite the importance of OAS in the coordination of S pools under stress, mechanisms of OAS perception and signaling have remained elusive. Here, we put particular focus on the general OAS-responsive genes but also elaborate on the specific roles of SDI1 and SDI2 genes, which downregulate the glucosinolate (GSL) pool size. We also highlight the key open questions in sulfur partitioning.

Metabolomic markers and physiological adaptations for high phosphate utilization efficiency in rice.

Utilizing phosphate more efficiently is crucial for sustainable crop production. Highly efficient rice (Oryza sativa) cultivars have been identified and this study aims to identify metabolic markers associated with P utilization efficiency (PUE). P deficiency generally reduced leaf P concentrations and CO2 assimilation rates but efficient cultivars were reducing leaf P concentrations further than inefficient ones while maintaining similar CO2 assimilation rates. Adaptive changes in carbon metabolism were detected but equally in efficient and inefficient cultivar groups. Groups furthermore did not differ with respect to partial substitutions of phospholipids by sulfo- and galactolipids. Metabolites significantly more abundant in the efficient group, such as sinapate, benzoate and glucoronate, were related to antioxidant defence and may help alleviating oxidative stress caused by P deficiency. Sugar alcohols ribitol and threitol were another marker metabolite for higher phosphate efficiency as were several amino acids, especially threonine. Since these metabolites are not known to be associated with P deficiency, they may provide novel clues for the selection of more P efficient genotypes. In conclusion, metabolite signatures detected here were not related to phosphate metabolism but rather helped P efficient lines to keep vital processes functional under the adverse conditions of P starvation.

Functional Features of TREHALOSE-6-PHOSPHATE SYNTHASE1, an Essential Enzyme in Arabidopsis.

In Arabidopsis (Arabidopsis thaliana), TREHALOSE-6-PHOSPHATE SYNTHASE1 (TPS1) catalyzes the synthesis of the sucrose-signaling metabolite trehalose 6-phosphate (Tre6P) and is essential for embryogenesis and normal postembryonic growth and development. To understand its molecular functions, we transformed the embryo-lethal tps1-1 null mutant with various forms of TPS1 and with a heterologous TPS (OtsA) from Escherichia coli, under the control of the TPS1 promoter, and tested for complementation. TPS1 protein localized predominantly in the phloem-loading zone and guard cells in leaves, root vasculature, and shoot apical meristem, implicating it in both local and systemic signaling of Suc status. The protein is targeted mainly to the nucleus. Restoring Tre6P synthesis was both necessary and sufficient to rescue the tps1-1 mutant through embryogenesis. However, postembryonic growth and the sucrose-Tre6P relationship were disrupted in some complementation lines. A point mutation (A119W) in the catalytic domain or truncating the C-terminal domain of TPS1 severely compromised growth. Despite having high Tre6P levels, these plants never flowered, possibly because Tre6P signaling was disrupted by two unidentified disaccharide-monophosphates that appeared in these plants. The noncatalytic domains of TPS1 ensure its targeting to the correct subcellular compartment and its catalytic fidelity and are required for appropriate signaling of Suc status by Tre6P.

Multifaceted regulatory function of tomato SlTAF1 in the response to salinity stress.

Salinity stress limits plant growth and has a major impact on agricultural productivity. Here, we identify NAC transcription factor SlTAF1 as a regulator of salt tolerance in cultivated tomato (Solanum lycopersicum). While overexpression of SlTAF1 improves salinity tolerance compared with wild-type, lowering SlTAF1 expression causes stronger salinity-induced damage. Under salt stress, shoots of SlTAF1 knockdown plants accumulate more toxic Na+ ions, while SlTAF1 overexpressors accumulate less ions, in accordance with an altered expression of the Na+ transporter genes SlHKT1;1 and SlHKT1;2. Furthermore, stomatal conductance and pore area are increased in SlTAF1 knockdown plants during salinity stress, but decreased in SlTAF1 overexpressors. We identified stress-related transcription factor, abscisic acid metabolism and defence-related genes as potential direct targets of SlTAF1, correlating it with reactive oxygen species scavenging capacity and changes in hormonal response. Salinity-induced changes in tricarboxylic acid cycle intermediates and amino acids are more pronounced in SlTAF1 knockdown than wild-type plants, but less so in SlTAF1 overexpressors. The osmoprotectant proline accumulates more in SlTAF1 overexpressors than knockdown plants. In summary, SlTAF1 controls the tomato's response to salinity stress by combating both osmotic stress and ion toxicity, highlighting this gene as a promising candidate for the future breeding of stress-tolerant crops.